In vivo visualization of angiogenesis during wound healing featured on journal cover

Innovative ways of imaging wound healing can reveal much about blood vessel remodeling and blood flow following an injury. Researchers in the Russell H. Morgan Department of Radiology and Radiological Science and Department of Biomedical Engineering at the Johns Hopkins University School of Medicine have developed a method for using laser speckle contrast imaging (LSCI) to elucidate the changes that occur in the microvasculature over time as a wound heals. Researchers in the laboratory of Arvind P. Pathak have visualized the wound healing process in a mouse ear model by capturing images of angiogenesis—or the development of new blood vessels—over a 12-day period.

“LSCI is a powerful tool for observing the architecture and remodeling of microvasculature as well as the hemodynamic changes (blood flow) during angiogenesis,” said Pathak, an assistant professor of radiology and oncology and principal investigator on the study. “Being able to watch this process occur in a living animal helps us better understand the role of the vasculature during various phases of the wound healing process.”

Stunning images obtained from their experiments were featured on the cover of the March issue of the journal Angiogenesis. The LSCI images shown on the cover from left to right are sequential images of the in vivo blood flow changes that occur on days 0, 3, 5, 7, 10 and 12 after wound creation. The “hotter” colors indicate higher blood flow. The background image is a grayscale LSCI image from an uninjured mouse ear.

Wound healing typically proceeds in three phases, Pathak explained: inflammation (which initiates the immune response and recruits immune cells and molecules to the injury), proliferation (the formation of new blood vessels and epithelium) and remodeling (which removes the vascular scar created during blood vessel formation). LSCI is ideal for imaging the progression of each phase because it can monitor in vivo changes in microvascular architecture and hemodynamics at the same time, he said.

LSCI images are created when tissue illuminated by a laser is photographed through a small aperture, explained Pathak. “The resulting images exhibit a random interference pattern, also called a ‘speckle’ pattern. In blood vessels, this speckle pattern shifts due to the orderly motion of red blood cells, causing a blur over the exposure time of the camera. The degree of blurring in the LSCI image is proportional to the velocity of blood in the vessels and constitutes the biophysical basis of LSCI. Therefore, LSCI can distinguish blood vessels in tissue without any fluorescent dye or contrast agent.”

In this way, Pathak added, LSCI is capable of “wide area mapping” of the tissue, allowing us to measure not only the length and perfusion of blood vessels but their tortuosity (twistiness) and the overall flow of blood to the wound site as healing progresses.

In addition to angiogenesis research, the imaging method has practical applications in drug testing, Pathak said. “Using LSCI alongside a drug study would provide better insight into the efficacy of drug delivery and therapeutic outcome,” he said.

The lead author of the paper was Abhishek Rege, a graduate student in biomedical engineering co-mentored by Pathak and Nitish V. Thakor, professor of biomedical engineering in whose neuroengineering laboratory LSCI was developed. Kevin Rhie, a research technician in Pathak’s laboratory was the other author on this study.

This work was supported jointly by a Johns Hopkins Institute for NanoBiotechnology (INBT) Junior Faculty Pilot Award to Pathak, and grants from the National Institute of Aging and the Department of Health and Human Services to Thakor.